Method for forming biochips and biochips with non-organic landings for improved thermal budget

ABSTRACT

The present disclosure provides biochips and methods of fabricating biochips. The method includes combining three portions: a transparent substrate, a first substrate with microfluidic channels therein, and a second substrate. Through-holes for inlet and outlet are formed in the transparent substrate or the second substrate. Various non-organic landings with support medium for bio-materials to attach are formed on the first substrate and the second substrate before they are combined. In other embodiments, the microfluidic channel is formed of an adhesion layer between a transparent substrate and a second substrate with landings on the substrates.

PRIORITY

This application is a continuation of U.S. patent application Ser. No.14/497,151, filed on Sep. 25, 2014, and entitled, “Method for FormingBiochips and Biochips With Non-Organic Landings for Improved ThermalBudget,” which is a divisional of U.S. patent application Ser. No.13/801,182, filed on Mar. 13, 2013, and entitled, “Method for FormingBiochips and Biochips With Non-Organic Landings for Improved ThermalBudget,” now U.S. Pat. No. 8,846,416, issued Sep. 30, 2014, whichapplications are incorporated herein by reference.

FIELD

This disclosure relates to biosensors and methods for forming bio-chips.Particularly, this disclosure relates to bio-chips having biosensors andfluidic devices and methods for forming them.

BACKGROUND

Biosensors are devices for sensing and detecting biomolecules andoperate on the basis of electronic, electrochemical, optical, and/ormechanical detection principles. Biosensors can sense charges, photons,and mechanical properties of bio-entities or biomolecules, or throughmolecular tags. The detection can be performed by detecting thebio-entities or biomolecules themselves, or through interaction andreaction between specified reactants and bio-entities/biomolecules. Suchbiosensors can be manufactured using semiconductor processes, and can beeasily applied to integrated circuits (ICs) and microelectromechanicalsystems (MEMS).

Biochips are essentially miniaturized laboratories that can performhundreds or thousands of simultaneous biochemical reactions, such aspolymerase-chain reactions (PCR) including solid phase/bridgeamplification. Biochips can detect particular biomolecules, measuretheir properties, process the signal, and may even analyze the datadirectly. Biochips enable researchers to quickly screen large numbers ofbiological analytes for a variety of purposes, from disease diagnosis todetection of bioterrorism agents. Advanced biochips use a number ofbiosensors along with fluidic channels to integrate reaction, sensing,and sample management. While biochips are advantageous in many respects,challenges in their fabrication and/or operation arise, for example, dueto compatibility issues between the semiconductor fabrication processes,the biological applications, and restrictions and/or limits on thesemiconductor fabrication processes.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the present disclosure are best understood from the followingdetailed description when read with the accompanying figures. It isemphasized that, in accordance with the standard practice in theindustry, various features are not drawn to scale. In fact, thedimensions of the various features may be arbitrarily increased orreduced for clarity of discussion.

FIG. 1A is a top view of a biochip in accordance with variousembodiments of the present disclosure.

FIG. 1B is a cross section view of a biochip in accordance with variousembodiments of the present disclosure.

FIG. 2 is a flow chart of various embodiments of methods of fabricatinga biochip device according to one or more aspects of the presentdisclosure.

FIGS. 3A-3H are cross-sectional views of a biochip in accordance withvarious embodiments according to methods of FIG. 2 of the presentdisclosure.

FIG. 4 is a flow chart of various embodiments of methods of fabricatinga biochip device according to one or more aspects of the presentdisclosure.

FIGS. 5A-5L are cross-sectional views of a biochip in accordance withvarious embodiments according to methods of FIG. 4 of the presentdisclosure.

FIGS. 6A and 6B are flow charts of various embodiments of methods offabricating a biochip device according to one or more aspects of thepresent disclosure.

FIGS. 7A-7G are cross-sectional views of a biochip in accordance withvarious embodiments according to methods of FIGS. 6A and 6B of thepresent disclosure.

FIG. 8 is a flow chart of various embodiments of methods of fabricatinga biochip device according to one or more aspects of the presentdisclosure.

FIGS. 9A-9G are cross-sectional views of a biochip in accordance withvarious embodiments according to methods of FIG. 8 of the presentdisclosure.

DETAILED DESCRIPTION

It is to be understood that the following disclosure provides manydifferent embodiments, or examples, for implementing different featuresof the invention. Specific examples of components and arrangements aredescribed below to simplify the present disclosure. These are, ofcourse, merely examples and are not intended to be limiting. Moreover,the formation of a first feature over or on a second feature in thedescription that follows may include embodiments in which the first andsecond features are formed in direct contact, and may also includeembodiments in which additional features may be formed interposing thefirst and second features, such that the first and second features maynot be in direct contact. Further still, references to relative termssuch as “top”, “front”, “bottom”, and “back” are used to provide arelative relationship between elements and are not intended to imply anyabsolute direction. Various features may be arbitrarily drawn indifferent scales for simplicity and clarity.

A simple conventional biochip involves various bioreceptors which reactwith various biological material of interest in one or more patternedsites. One such reaction is the polymerase chain reaction (PCR) that canmultiply by orders or magnitude the number of molecular strands at asite. Various approaches are used to differentiate among differentreactants and reactions for detection. One common approach is to tag areaction with a fluorescent or phosphorescent label that emits adetectible photon. A coordinated, or ordered array, approach wouldencode the sensor or bioreceptors to a location on the biochip, so thata positive reaction and photo-detection would be correlated to thelocation to determine the nature of the reaction, for example, identityof the biological material. In many cases, the location is externallyobserved by optical detection. In other cases, the location correspondsto embedded sensors that signal a measurement. The signal may beoptical, magnetic, electrical, or a mass-sensitive measurement such assurface acoustic wave or microbalance weights. Another approach is therandom approach that encodes the sensor with different fluorescence,phosphorescence, or otherwise detectible and differentiable tags. Apositive detection would be correlated to the type of signal transducedto determine the nature of the detection. The signal transduced may bephotons, for example, where a different wavelength of light is generatedfor different biological materials or reactions. In another example,surface plasmon resonance could be used to detect different biologicalmaterials without the use of fluorescent or phosphorescent tags orlabels.

More advanced biochips involve not only biosensors, but also variousfluidic channels to deliver biological material to the sensors. Thefluidic channels may be a part of a microfluidic system that includespumps, valves, and various measurement devices such as flow meters,pressure transducers, and temperature sensors. Combination of fluidprocessing and sensing is advantageously integrated within asemiconductor chip environment. A potential use of biochips is as alab-on-a-chip—where medical professionals can use a small biochip toperform testing in the field, obtain results directly, and proceed withtreatment or further analysis without retreating to a laboratory.Especially for medical professionals working in remote areas wheresample preservation may be difficult, lab-on-a-chip devices can savelots of traveling and waiting. These lab-on-a-chip devices are oftensingle-use, or disposable, devices. As such, the manufacturing costshave to be low to be economically viable.

Semiconductor processing often involves baking, curing, and exposingvarious surfaces to plasma energy and radiation energy. At hightemperatures (i.e., above about 75 degrees Celsius, above 100 degreesCelsius, or over 150 degree Celsius) and/or high energies, theseprocesses would damage or destroy organic bioreceptors and surfacemodification layers, which usually are delicate bio-molecules or verythin layers of surface chemistry. For example, the bioreceptors may beantibodies/antigens, enzymes, nucleic acids/DNA, cellularstructures/cells, and biomimetic receptors. The surface modificationchemistry may include layers of hexamethyldisilazane (HMDS),3-aminopropyl triethoxysilane (APTES), agar, or hydrogel.

Thus, the bio-functionalization of surfaces on which bio-molecules areattached, are often performed after all the semiconductor processes arecompleted to avoid being exposed to the high temperature processes. Insome designs, the fluidic channels are formed directly on thesemiconductor substrate, usually silicon wafer and hereinafter referredto as the sensing wafer, along with bioreceptor sites, hereinafterreferred to as landings. In other designs, the fluidic channels areformed on a fluidic substrate that is subsequently bonded to the sensingwafer having the landings. In some designs, the biosensors are embeddedand integrated with the landings. In other designs, the biosensors arenot intrinsically integrated with the landings. In such case, thefluidic channel has a transparent lid, which allows an external opticaldetection of biomolecules or reactions. In the case where the fluidicchannel is on the sensing wafer, the fluidic channel formation, usuallyetching a trench or via into the substrate, can damage the biosensors,bioreceptors, or surface modification layer. To avoid this damage, whena high temperature bonding process is used, the bioreceptors aredeposited on the interior walls of the fluidic channels after thebonding process and the fluidic channels are enclosed, usually byflowing through each biochip a high concentration of bioreceptorsthrough the fluidic channel surfaces having some affinity for thebioreceptors. However, the density of bioreceptors that attach to thesurfaces is hard to control, and the process is slow and wasteful ofbioreceptors and reagents. In some cases, the bound-bioreceptor densityvaries throughout the biochip or a batch of biochips (not uniform) asthe concentrations in the reagents change. The random, non-alignedlocations and non-uniform concentrations complicate resolution ofdetectible activities at different sites using image processingalgorithms. The locations may overlap each other and are hard toresolve. The randomness also makes difficult correlations betweendifferent biochips because each would have different bioreceptormapping.

The various embodiments of the present disclosure contemplates awafer-level process and a biochip that addresses many of these issues byproviding non-organic landings that are resistant to high temperatureprocessing as bioreceptors sites. The bioreceptors, for example, DNAprimers, are attached to the landings after the biochip is fabricated.The site locations are patterned with an adhesive layer and a supportmedium to form the landings. The density issue as well as the random,non-aligned location issue are addressed by forcing the bioreceptors toattach only at the landing sites. The use of high-temperature resistancelandings allow certain semiconductor processes to be used in fabricatingthe biochip that otherwise cannot be used. The various methodembodiments of the present disclosure may be performed in asemiconductor fabrication facility and can be configured to becompatible with the complementary metal-oxide-semiconductor (CMOS)process. In more detail, the processing of materials on glass andetching of glass are often incompatible with some stages of the CMOSprocess because the glass processing can introduce particles that areconsidered contaminants for other CMOS processes. Some embodiments ofthe present disclosure involve no glass processing or minimal processingof glass when it is used as a transparent substrate. In someembodiments, the glass processing is performed separately from the otherprocesses to avoid introduction of contaminants.

In certain embodiments, the biochip of the present disclosure is formedby combining three substrates, at least one of which is transparent.FIG. 1A is a top view of a biochip 100 in accordance with someembodiments of the present disclosure. FIG. 1B is a cross-section of thebiochip 100 from sectional line A-A′ of FIG. 1A. The top view of FIG. 1Ais cut from sectional line B-B′ of FIG. 1B. Biochip 100 includes a firstsubstrate 101 bonded to a transparent substrate 103 and a secondsubstrate 105. The second substrate 105 has a fluidic inlet 107/109 anda fluidic outlet 109/107 through the second substrate 105. The firstsubstrate 101 includes microfluidic channel patterns, shown as channels111, 113, and 115. The various channels 111, 113, and 115 are connectedto each other via various pathways and may be different sizes dependingon the design of the biochip. The channels include various landings ontop of the channels close to the transparent substrate 103 or bottom ofthe channels on the second substrate 105, or both. FIG. 1B shows thelandings on both top and bottom of the channels. First landings 117 areformed on the first substrate 101 and surrounded by a passivating layer121. Second landings 119 are formed on the second substrate 105. Thefirst and second landings may have different densities in differentchannels. The landings have certain chemistries that allow some materialto bind to it. In some cases, the landings may provide a hydrophilic, ahydrophobic, or another surface chemistry such as affinity forparticular functional groups. According to various embodiments, asupport medium on which various bio-materials can bind, including agaror polyethylene glycol (PEG) hydrogel, is disposed on the landings. Thesupport medium is connected to the landing on the substrate through anadhesion layer, which may be 3-aminopropyl triethoxysilane (APTES), orhexamethyldisilazane (HMDS). The first landing 117 and second landings119 on the substrate may be different materials to provide differentchemistries. In some cases, the landings on the substrate are siliconoxide, certain metal oxides, or metal.

An oxide layer 123 covers the passivating layer 121 and is bonded to thetransparent substrate 103. The first substrate 101 and the transparentsubstrate 103 are bound by an oxide-oxide binding process. The firstsubstrate 101 and the second substrate 105 are bound by asilicon-silicon binding process. Very good adhesion is achieved withoutany chance of leakage for these bonds because no adhesive is used.

FIG. 2 is a flow chart of some embodiments of methods 200 of fabricatinga biochip device according to one or more aspects of the presentdisclosure. FIGS. 3A to 3H are cross-sectional views of partiallyfabricated biochip devices constructed according to one or more steps ofthe method 200 of FIG. 2. In operation 201 of FIG. 2, a number of firstlandings is formed on a first substrate. FIG. 3A shows the firstsubstrate 301. The first substrate 301 may be silicon, sapphire, siliconcarbide, or other commonly used semiconductor substrates that does notreact with the analyte or solution. In some embodiments, the firstlandings are formed by depositing an oxide layer and patterning theoxide layer. The oxide layer may be between about 100 nm and about 200nm thick. The oxide layer may be doped or undoped silicon oxide or ametal oxide. The oxide layer pattern including first landings 303 andoxide blocks 305. The transparent substrate is subsequently attached tothe first substrate through the oxide blocks 305.

In some embodiments, the first landings are not oxides. The firstlandings may be metal, for example gold or platinum that showsparticular affinity for certain chemical groups or proteins. Metallicfirst landings are formed by depositing a layer of metal and patterningthe metal layer by etching or depositing a patterned mask layer beforedepositing the metal layer, then removing the mask layer along withoverlying metal in a lift-off operation.

Referring back to FIG. 2, in operation 202, a passivating layer isdeposited over and between the plurality of first landings on the firstsubstrate. FIG. 3B shows a first substrate 301 with a passivating layer307 over the first landings 303 and oxide blocks 305. The passivatinglayer 307 has a surface which does not attract or bind a biomaterialintended for a landing site. The passivating layer 307 may be siliconnitride at about 700 nm.

Referring back to FIG. 2, in operation 203 an oxide layer is depositedand planarized over the passivating layer on the first substrate. FIG.3B shows the oxide layer 309 over the passivating layer 307. The oxidelayer 309 may include the same material or different material from thefirst landing 303. After planarization, a thickness of at least onehundred nanometers (nm) remains. According to some embodiments, theoxide layer 309 is between about 300 and about 500 nm. The oxide layer309 is planarized to ensure a smooth and flat surface for fusion bondingwith a transparent substrate in operation 205 of FIG. 2. The transparentsubstrate may be glass, quartz, sapphire, or other transparentsubstrate. The oxide to glass bond may involve water between the oxideand glass and anneal at a temperature above 150 to 300 degrees Celsius.The glass substrate may be about 500 microns or thicker. FIG. 3C shows atransparent substrate 311 bonded to the oxide layer 309.

Referring back to FIG. 2, in operation 207 a backside of the firstsubstrate is etched to expose at least some of the first landings. Theopenings are the microfluidic channels of the biochip. The firstsubstrate may be thinned first by grinding to less than about 150microns, or about 100 microns or less. A mask pattern is used to etchthe first substrate from the backside and stop on the first landing andpassivating layers. In some embodiments, a wet etch involves potassiumhydroxide (KOH), tetramethylammonium hydroxide (TMAH), or ethylenediamine and pyrocatechol (EDP). The selection of different etchantinvolves the type amount of silicon to be etched, as the etch ratesdiffer, and the thickness of first landing and passivating layers. Athick first landing, for example, about 200 nm or greater, allows someover etching to occur without risk to removing the first landingaltogether. The silicon plane to be etched affects the etch selectivityamong different etchants. One skilled in the art would choose the wetetch process appropriate for the situation. In other embodiments, a dryetch involving fluorine or chlorine-containing plasma is used through amask pattern. The dry etch results in less undercutting of the siliconunder the etch mask. However, a small amount of over etching of thefirst landing and passivating layers is expected. FIG. 3D is the crosssection of a partially fabricated biochip with microfluidic channels313, 315, and 317 formed by etching into first substrate 301. The firstlandings 303 and portions of the passivating layer 307 are exposed inthe bottom of the microfluidic channels 313, 315, and 317.

Referring back to FIG. 2, in operation 209 a plurality of secondlandings are formed on a second substrate. The second substrate may besilicon, sapphire, silicon carbide, or other commonly used semiconductorsubstrates that does not react with the analyte or solution. Thedifferent ways to form the second landings are the same as the waysdescribed to form the first landings in association with operation 201.In short, a layer is either patterned by etching or deposited into apattern and lifted-off. The second landings may be of a same material asthe first landing or different material and the formation process may besame or different. FIG. 3E shows the second substrate 351 having secondlandings 353 and blocks 355 thereon.

The second substrate may be a sensing wafer containing sensing andmicrofluidic machines for fluid processing. Sensors such as temperaturesensor, pressure transducer, and flow meter may be fabricated first onthe sensing wafer along with microfluidic machines such as pumps andvalves. In some embodiments, the sensing wafer includes electrodes andmagnets for directing fluid flow and for separating certain componentsof the analyte. The sensing wafer may also include localized heaters ateach landing or for a group of landings. Separate controls for thelocalized heaters allow different amplification of bio-materials fortesting. The heater may be embedded in the substrate under the landingor disposed on the substrate surface close to the landing.

The blocks 355 may be removed by wet etching in a separate, optionaloperation to expose a fusion bonding area under the blocks. Ifperformed, a separate etch mask is used to perform the wet etching. Thisoperation may be performed if the blocks on the second substrate surfaceafter patterning in operation 209 cannot be used directly for fusionbonding. As understood, fusion bonding can have defects and create voidsbetween the substrates bonded if the substrate surface is not flat andparticle-free. The blocks on the second substrate may contain etchresidues not favorable for fusion bonding. Wet etching can completelyremove the block 355 to form a second substrate surface suitable forfusion bonding. FIG. 3F shows the second substrate 351 with only thesecond landings 353 thereon after the blocks 355 have been removed.

Referring to FIG. 2, in operation 211 a protective layer is depositedover the plurality of second landings on the second substrate. Theprotective layer is material layer that separates any laser drillingbyproduct from the substrate and the landings. The protective layer iseasily removed from the substrate. According to various embodiments, theprotective layer is Skycoat or Nanoshelter, both available from NikkaSeiko Co., Ltd of Tokyo, Japan. In other examples, the protective layeris silicon nitride or water-soluble wax. The protective layer isgenerally deposited in a fluid phase, either liquid or vapor, onto thesecond substrate surface. In some embodiments, the protective layer iswater soluble and is cleaned away along with any deposits thereoneasily. In other embodiments, the protective layer reacts with anotherchemical to form a gas and any deposits thereon can be vacuumed away. Asilicon nitride protective layer is removed using a hot phosphoric acidbath.

Referring to FIG. 2, in operation 213, through-holes are formed in thesecond substrate. In some embodiments, the through-holes are formed bylaser drilling, microblasting, or ultrasonic drilling. Other techniquesof forming through-holes include various etching techniques and waterjetdrilling. Laser drilling of cylindrical holes generally occurs throughmelting and vaporization (also referred to as “ablation”) of thesubstrate material through absorption of energy from a focused laserbeam. Depending on the direction of the laser energy, the laser drilledthrough-holes can have the inverse trapezoidal shape in a cross section.Microblasting removes material by driving a high velocity fluid streamof air or inert gases of fine abrasive particles, usually about 0.001 in(0.025 mm) in diameter. Ultrasonic drilling involves using highfrequency vibrations to hammer a bit through materials. At least twothrough-holes are formed for every biochip—an inlet and an outlet. Morethan two through-holes may be used for different inlet fluids or if thebiochip performs separation of the analyte and more than one outlet isused. Depending on the process used for forming the through-holes, byproducts of the drilling may be attached to the protective layer. FIG.3G is a cross sectional diagram of the second substrate 351 having aprotective layer 357 over the second landings 353 and two through-holes359 and 361.

Referring to FIG. 2, in operation 215, the protective layer is removed.As discussed in association with operation 211, the protective layerremoval depends on the type of protective layer used. The protectivelayer may be removed by rinsing, steam cleaning, wet etching, andexposing the substrate to particular chemical vapors, heat, orradiation. Any deposits on the protective layer from the drillingoperation are removed along with the protective layer. FIG. 3H is across sectional diagram of the second substrate 351 having secondlandings 353 and two through-holes 359 and 361.

Referring to FIG. 2, in operation 217, the first substrate and thesecond substrate are fusion bonded. The fusion bonding attaches thefirst substrate of FIG. 3D to second substrate of FIG. 3H to form theworkpiece of FIG. 1B. According to various embodiments, the firstsubstrate is silicon and the second substrate is also silicon. Asilicon-to-silicon fusion bond involves bonding two silicon surfacesbased on intermolecular interactions including van der Waals forces,hydrogen bonds, and strong covalent bonds. The two substrates arealigned and moved close together. If the surfaces are sufficientlysmooth, the substrates start to bond as soon as they get in atomiccontact. If the surface is activated by plasma to form silanol groups,at room temperature a significant fraction of Si—OH (silanol) groupsstart to form Si—O—Si and water. The formed water molecules will migrateor diffuse along the interface during a subsequent annealing process. Insome circumstances, the substrates may be covered with water moleculesso the bonding happens between chemisorbed water molecules on theopposing substrates surfaces. According to various embodiments, methodsto enhance fusion bonding include plasma, ultra high vacuum, andchemical-mechanical polishing (CMP).

In optional operation 219, the first substrate and the second substrateare annealed. The anneal may be optional depending on the surfacetreatment and type of silicon surface bonded. A post-bond anneal isabove 300 degree Celsius and may be over 700 degree Celsius. At over 300degrees Celsius, covalent Si—Si bonds start to establish between the twosubstrate surfaces. At 700 degrees Celsius, newly established Si—Sibonds can reach cohesive strengths of bulk silicon. The annealingtemperature depends on desirable bond strength and differentpretreatments. In one example, one or more substrate is exposed to aplasma, fusion bonded, and then annealed at about 500 to about 800degrees Celsius.

Referring to FIG. 2, in operation 221 the transparent substrate isgrinded. In one-example, the transparent substrate is grounded to about300 microns. The transparent substrate may be glass. The grindingprocess stresses the oxide-oxide bond between the transparent substrateand the first substrate and the silicon-silicon bond between the firstsubstrate and the second substrate and may separate these substrates ifdefects and voids reduce the bonding strength.

In optional operation 223, adhesion, support medium, and bio-materialsare flowed separately through the microfluidic channels in the firstsubstrate. The adhesion, support medium, and bio-materials enter themicrofluidic channels through one of the through-holes through thesecond substrate and exit the microfluidic channels through the otherthrough-holes in the second substrate. Referring to FIG. 1A, in someembodiments, the adhesion, support medium, and bio-materials enter thebiochip at through-hole 107 and flows through channels 111, 113, and 115before exiting the biochip at through-hole 109. The various landings 117and 119 are exposed to the adhesion, support medium, and bio-materials.The flow may be in vapor or liquid phase. As the adhesion, supportmedium, and bio-material flows through the channels, the materialssequentially adhere to the landing sites, which may be the firstlandings or the second landings, and avoid other regions, for example,the protective layer 121 and inner surface of the first substrate 101and second substrate 105.

The adhesion material may be 3-aminopropyl triethoxysilane (APTES) orhexamethyldisilazane (HMDS). For example, APTES selectively adheres onlyto the first and second landing features that are hydrophilic to form anadhesion layer. In a second flow, a support media material is flowedthrough the channels and adheres to the adhesion layer to form thesupport medium. The support media material may be agar or hydrogel, forexample, polyethylene glycol (PEG) hydrogel. In a third flow, shortpieces of single stranded DNA known as primers are flowed through andattached to the support medium. In a subsequent flow, longer singlestranded DNA are flowed through to hybridize with the primers. Accordingto some embodiments, these longer strands can be amplified in populationusing various PCR techniques at the landing to form clusters. Some ofthe strands may have fluorescent tags that become activated by thereaction. When exposed to light, the fluorescent tags produce a lightresponse that can be detected through the transparent substrate. Anoptical detection mechanism can detect the wavelength and intensity oflight to determine the extent of reaction and population of the DNAstrands.

After populating the landings with selected bio-materials, the biochipis used in the field or laboratory. In one example, the biochip is usedto test for the presence of certain DNA strands. While FIG. 1A shows asimple flow path for the bio-material, the present disclosure alsoenvisions much more complicated flow paths where some bio-material maybe lysed, separated, dyed, and then tested or analyzed using chemical,electrical, or optical means. For example, a drop of blood may beinserted in an inlet and initially separated by plasma and cell type.Certain cells in the blood drop may be lysed. Some macromolecules in thelysate may be further broken down for analysis by downstream in the flowpath. When more than one inlet and outlet through-holes are used,different bio-materials having different chemistries may attach tospecific or corresponding landing sites.

FIGS. 1A-1B, 2, 3A-3H, and associated text pertains to embodiments wherethe biochip inlet and outlet through-holes are located in the secondsubstrate, which is on the opposite of the transparent substrate. Incertain embodiments, the biochip inlet and outlet through-holes arelocated in the transparent substrate instead of the second substrate.The orientation of the optical detection determines the side of thetransparent substrate to allow external observation of internalreactions. FIGS. 4 and 5A-5L show the embodiments where thethrough-holes are in the transparent substrate. In still otherembodiments, the one of the through-holes, for example, the inlet, is ontop, and the other through-hole, for example, the outlet, is on thebottom. The transparent substrate may be on either side. FIG. 4 shows amethod 400 for forming a biochip in accordance with various embodimentsof the present disclosure. FIGS. 5A to 5L are cross sectional diagramsof partially fabricated biochips after various operations of method 400.Because some of the operations of method 400 are very similar or thesame as the operations of method 200, the similarities are merelyreferenced and not discussed in detail and differences are emphasized.In operation 401 of method 400 in FIG. 4, a number of first landings areformed on a first substrate. Operation 401 is the same as operation 201of FIG. 2. FIG. 5A shows the first substrate 501 having first landings503 and oxide blocks 505.

In operation 403, a passivating layer is deposited over and between thefirst landings on the first substrate. Operation 403 is the same asoperation 202 of FIG. 2. FIG. 5B shows the first substrate 501 havingfirst landings 503, oxide blocks 505, and a passivating layer 507deposited over and between the first landings. Next, in operation 404,an oxide layer is deposited and planarized over the passivating layer onthe first substrate. Operation 404 is the same as operation 203 of FIG.2. FIG. 5B shows the oxide layer 509 over the passivating layer 507.

Referring back to FIG. 4, in operation 405 the oxide layer on the firstsubstrate is fusion bonded to a transparent substrate. Operation 405 isthe same as operation 205 of FIG. 2. After the fusion bonding, thetransparent substrate is grinded in operation 407. The grindingoperation 407 is similar to operation 221 of FIG. 2; however, thegrinding operation 407 is performed before the second substrate isattached. A transparent substrate may be about 500 microns or thickerand is grinded to less than about 200 microns, or about 175 microns.FIG. 5C shows a first substrate 501 fusion bonded to a thinnedtransparent substrate 511.

In operation 408 of FIG. 4, the transparent substrate is bonded to acarrier wafer. The carrier wafer allows further processing on thebackside of the first substrate. The carrier wafer may be glass, arecycled silicon wafer, or another commonly used and recyclable carrierwafer. The bonding between the transparent substrate and carrier waferis a temporary bond. The temporary bond is strong enough to withstandgrinding of the first substrate and can be removed relatively easily. Insome embodiments, an ultraviolet (UV) sensitive adhesive is used betweenthe carrier wafer and the transparent substrate. The adhesive breaksdown upon UV exposure. In other embodiments, the adhesion readilydissolves upon exposure to certain chemicals, while the certainchemicals do not affect other portions of the biochip. FIG. 5D shows thecarrier wafer 512 attached to the transparent substrate 511, which isattached to the first substrate 501.

Referring back to FIG. 4, in operation 409 a backside of the firstsubstrate is etched to expose at least some of the first landings.Operation 409 is the same as operation 207 of FIG. 2. The firstsubstrate may be thinned first by grinding to less than about 150microns, or about 100 microns or less. FIG. 5E is the cross section of apartially fabricated biochip with microfluidic channels 513, 515, and517 formed by etching into first substrate 501. The first landings 503and portions of the passivating layer 507 are exposed in the bottom ofthe microfluidic channels 513, 515, and 517.

Referring back to FIG. 4, in operation 411 a protective layer isdeposited over the first landings on the first substrate. The operation411 is similar to operation 211 of FIG. 2, but the protective layer isapplied to a different substrate. In operation 411, the protective layeris applied to a backside of the first substrate having microfluidicchannels that exposes the first landings. The protective layer ensuresthat by products from a drilling process would not affect the firstsubstrate surface quality.

In operation 413, through-holes are formed in the transparent substratethrough the protective layer, passivating layer, and the oxide layer.While through-holes are also formed in operation 213, the substrates aredifferent and different process parameters apply. In operation 413, thethrough-hole is formed by laser drilling or ultrasonic drilling. A lasercan focus its beam at a specific depth and do not puncture through thecarrier wafer. On the other hand, other drilling methods, such asmicroblasting, may damage the carrier wafer and render it unrecyclable.In some embodiments, ultrasonic drilling is used. If the adhesivebetween the carrier wafer and the first substrate can sufficientlydampen the ultrasound such that the carrier wafer is not damaged, thenultrasonic drilling may be used. In other embodiments, the carrier waferis not recycled and may be used to absorb any excess energy from thedrilling operation. One skilled in the art would adjust the process toform the through-holes in cross section diagram of FIG. 5F showing thefirst substrate 501 having a protective layer 557 over the firstlandings 503 and two through-holes 559 and 561.

Referring back to FIG. 4, in operation 414 the protective layer isremoved. Operation 414 is the same as operation 215 of FIG. 2. FIG. 5Gis a cross section of the partially fabricated biochip without theprotective layer. The partially fabricated biochip includes the firstsubstrate 501, having microfluidic channels 513, 515, and 517 andthrough-holes 559 and 561 in the transparent substrate 511 but notthrough the carrier wafer 512.

Referring back to FIG. 4, in operation 415 a second substrate havingsecond landings is provided. If the second landings are formed by thesame entity that formed the first landings, then operation 415 is thesame as operation 209 of FIG. 2. FIG. 5H shows the second substrate 551having second landings 553 and blocks 555 thereon. In other embodiments,second substrates are provided by a different entity that forms thesecond landings. The second landings may or may not be the same materialas the first landings. Just as with method 200, the blocks 555 may notbe formed or is removed by wet etching to expose a fusion bonding areaunder the blocks 555. The removal operation is the same as thatdescribed in association with operation 209 of FIG. 2. FIG. 5I shows thesecond substrate 551 with only the second landings 553 thereon after theblocks 555 have been removed.

Referring back to FIG. 4, in operation 417, the first substrate and thesecond substrate are fusion bonded. This fusion bonding operation 417 issimilar to operation 217 of FIG. 2. The location of through-holes andthe presence of carrier wafer may change the fusion bonding process.Operation 417 is preferably completed in a vacuum environment. When thefirst substrate and the second substrate come into contact, themicrofluidic channels are sealed within the biochip because thethrough-holes are covered by the carrier wafer. When the fusion bondingis completed at ambient pressure, air may be trapped under pressure andcan cause the substrates to separate if heated. FIG. 5J shows the bondedsubstrates (workpiece), with second substrate 551 at one side, firstsubstrate 501 adjoining the second substrate 551, transparent substrate511 next to the first substrate 501, and carrier wafer 512 on theopposite side from the second substrate 551.

Referring back to FIG. 4, in operation 419, the first substrate, secondsubstrate, transparent substrate, and carrier wafer are annealed. Theanneal operation 419 is the same as operation 219 of FIG. 2, with theaddition of carrier wafer in the anneal. As long as the varioussubstrates have similar coefficients of thermal expansion (CTEs), theaddition of carrier wafer does not affect the anneal process.

In operation 421, the carrier wafer is removed. The carrier wafer may beremoved by reacting the adhesive chemically or optically, or bydecomposing the interface layer. In some embodiments, the workpiece isexposed to a vapor or liquid that reacts or dissolves the adhesive. Inother embodiments, the adhesive is exposed to a radiation that breaks itdown chemically, for example, an UV light. In still other embodiments, ashort burst of laser focused at the interface between the carrier waferand the transparent substrate can be used to de-bond the two. Regardlessof method, care must be taken to not damage the transparent substratesurface. While the transparent substrate surface can be polished ifdamaged, the through-hole openings on the transparent substrate wouldhave to be plugged to avoid damage inside the microfluidic channels.

In some embodiments, operations 419 and 421 may be switched. If thefusion bonding without anneal has a high enough bond strength towithstand the carrier wafer de-bonding, then the carrier wafer may beremoved first. Switching the order of operations 419 and 421 may beperformed especially when the adhesive between the carrier wafer and thetransparent substrate would harden during the anneal. FIG. 5K shows thepartially fabricated biochip after the carrier wafer has been removed.

Referring back to FIG. 4 in operation 423, bio-materials may be flownthrough the microfluidic channels in the first substrate. Operation 423is the same as operation 223 of FIG. 2. FIG. 5L shows the biochip 500having a second substrate 551, a first substrate 501 bonded to thesecond substrate 551, and a transparent substrate 511 bonded to thefirst substrate 501. Microfluidic channels 513, 515, and 517 are formedthrough the first substrate 501, which is the sidewalls. Through-holes559 and 561 connect to the microfluidic channels through the transparentsubstrate 511 and portions of an oxide layer 509 and a passivating layer507 on the first substrate 501. In a top view, the microfluidic channels513, 515, and 517 connect to each other. The microfluidic channelsinclude first landings 503 and second landings 553 on opposite sides.The first landings 503 are on the side of transparent substrate 511 andexposed by etching away the first substrate under the first landings503. The passivating layer 507 surrounds the first landings. An oxidelayer 509 bonds the transparent substrate 511 to the first substrate501. The bio-materials 519 adhere to the first and second landings503/553 and can be used to bio-functionalize the biochip.

The following embodiments are processes of forming biochips and biochipsthat do not include a first substrate as the microfluidic channelstructure. Instead, the sidewalls of the microfluidic channel structureare formed by depositing and patterning another material. FIGS. 6A/6Band 7A to 7G show embodiments where the through-holes are in the secondsubstrate. FIGS. 8 and 9A to 9G show embodiments where the through-holesare in the transparent substrate. Because some of the operations ofthese embodiments are very similar or the same as the operations ofembodiments employing a first substrate, the similarities are merelyreferenced and not discussed in detail and differences are emphasized.

In operation 601 of method 600 in FIG. 6A, a transparent substrate isprovided. The transparent substrate may be glass, quartz, sapphire, orother transparent substrate. In operation 603 a carrier wafer is bondedto the transparent substrate. The bonding operation 603 is the same asoperation 408 of FIG. 4, except that the transparent substrate here is ablank substrate with no patterned structures thereon. FIG. 7A shows thetransparent substrate 701 bonded to a carrier wafer 703.

In operation 604, a patterned passivating layer is formed on thetransparent substrate. The first passivating layer may be a siliconnitride pattern formed by depositing a layer of silicon nitride andpatterning the silicon nitride. The patterning including depositing aphotoresist, exposing a light pattern on the photoresist, developing thephotoresist, using the remaining photoresist to etch the siliconnitride, and removing the remaining photoresist. The exposed transparentsubstrate between the silicon nitride patterns is the landing forbio-materials. FIG. 7B shows a transparent substrate 701 with apatterned passivating layer 705 thereon.

FIG. 6B is a process flow diagram of operation 604 in some embodiments.In operation 651, a silicon nitride layer is deposited on thetransparent substrate. The silicon nitride may be deposited using one ofchemical vapor deposition (CVD) techniques. In operation 653, thesilicon nitride layer is patterned. The patterning may be performedusing lithographic techniques. In operation 655 a silicon oxide layer isdeposited over the patterned silicon nitride layer. The silicon oxidefills the area between the silicon nitride patterns as well as over thesilicon nitride pattern. In operation 657, the silicon oxide layer isplanarized to expose the patterned silicon nitride layer. In otherwords, the silicon oxide layer deposited over the patterned siliconnitride is removed. The patterned silicon nitride is the passivatinglayer. The silicon oxide is the landing sites. FIG. 7C shows atransparent substrate 701 having a patterned silicon nitride 705 andsilicon oxide landing 707. The patterned silicon nitride 705 and thesilicon oxide landing 707 have a planar top surface.

Referring back to FIG. 6A, in operation 605 a patterned adhesion layeris formed on the transparent substrate. The patterned adhesion layer maybe any bio-compatible adhesive, glue, polymer, epoxy, bonder, or solderthat can provide a hermetic seal to form channels and wells. Thematerial may be a photoresist, a silicone, a thermoplastic, or variousinsulators with added adhesion material. The adhesion layer, afterpatterning, is the sidewalls of the microfluidic channels. According tosome embodiments, the depth or height of the sidewalls may be as much as100 microns or greater. The adhesion layer is compatible with CMOSprocesses and may be easily patterned. Photoresist material can bedesigned to have different surface viscosities and use differentdeposition process parameters to form a relatively uniform film over thesubstrate and can be a suitable adhesion layer. One such example isSU-8. Another example is poly(phenylmethyl)silsesquioxane (PSQ).Silicone material may be molded and shaped to various shapes and sizes.Once hardened, silicone material can provide a hydrophobic orhydrophilic surface as fluid channels. In some embodiments, a siliconematerial polydimethylsiloxane (PDMS) is patterned by a soft lithographyprocess. The PDMS is poured over a wafer having desired sidewall shapesetched into it and hardened. The PDMS is then removed and sealed to aglass substrate by activating the PDMS surface using RF plasma. In otherembodiments, the PDMS is molded using compression molding or injectionmolding directly on the first substrate. Thermoplastic materials canalso be patterned using soft lithography or molding processes. Suitablethermoplastic material includes poly(methyl methacrylate) (PMMA),polycarbonate (PC), and polyimide (PI). Various insulators may bedeposited and patterned using semiconductor processes. For example,silicon oxide, silicon nitride, or various commonly used insulators insemiconductor processing may be used. The sidewall structure materialmay have more than one layers. In some embodiments, the first layer addsthickness while the second or subsequent layers add adhesion propertiesand/or surface chemistry. FIG. 7D is a cross section diagram including apatterned adhesion layer 709 over the patterned silicon nitride 705 andthe silicon oxide landing 707.

Referring back to FIG. 6A, in operation 607 a second substrate havingsecond landings and a plurality of through-holes is provided. Suchsecond substrate and formation has been described with respect to themethod embodiments of FIG. 2 and FIG. 4. For example, the operations209, 211, 213, and 215 of method 200 results in a second substratehaving second landings and a plurality of through-holes, as shown inFIG. 3H. In other embodiments, the second substrate is formed by adifferent entity and the second substrate is provided. FIG. 7E shows thesecond substrate 711 with second landings 713 thereon.

Referring back to FIG. 6A, in operation 609 the transparent substrate isbonded to the second substrate via the patterned adhesion layer.Depending on the type of adhesion layer, it may be activated firstbefore bonding. Activation may include exposure to plasma, gas/vapors,fluid, heat, or radiation. Because the energy-sensitive bio-material hasnot been introduced, the patterned adhesion layer activation is notlimited to low energy methods. In the example of PSQ, an oxygen plasmabreaks bonds on the PSQ surface and creates dangling bonds which readilyadhere to a silicon oxide, a thin layer of which is always present on asilicon wafer exposed to ambient conditions. In the example of APTES,water catalyzes covalent bonds between APTES and a silicon-containingsubstrate at room temperature. The second substrate and the transparentsubstrate are aligned and brought into proximity of each other duringthe bonding process. Mechanical pressure may be applied to one or moreof the substrates to ensure good contact. The bonding process mayinvolve specific vacuum and temperature parameters. After the initialbonding, additional steps may be taken to strengthen the bond such ascuring under high temperature and exposure to certain radiation.

The carrier wafer is removed in operation 611 of FIG. 6A. The carrierwafer is removed by the process described in association with operation421 of method 400 in FIG. 4. FIG. 7F shows the second substrate 711bonded to the transparent substrate 701 via the patterned adhesion layer709. The carrier wafer is removed.

Referring back to FIG. 6A, in operation 613, support media material andbio-materials may be flown through the microfluidic channels in thefirst substrate. Operation 613 is the same as operation 223 of FIG. 2.FIG. 7G shows bio-material 715 attached to the first landings 707 andsecond landings 713.

In other embodiments, FIGS. 8 and 9A to 9G show methods and structurewhere the through-holes are in the transparent substrate instead of thesecond substrate. These embodiments involve various process operationsthat are already discussed in relation to different embodiments.Operations 801 and 803are the same as operations 601 and 603 of FIG. 6A.In operation 804, a patterned first passivating layer and first landingsare formed on the transparent substrate. Operation 804 is similar tooperations 651 to 657 as discussed in association with FIG. 6B. FIGS.9A, 9B, and 9C are the same as FIGS. 7A, 7B, and 7C. In operation 805,through-holes are formed in the transparent substrate. Operation 805 issimilar to operation 413 and associated FIG. 9D is similar to FIG. 5Fthat is associated with operation 413. The transparent substrate ofoperation 413 has a patterned passivating layer and first landings andis bonded to a first substrate.

Referring back to FIG. 8, in operation 807, second landings are formedon the second substrate. Operation 807 is the same as operation 209 ofFIG. 2. In operation 808, patterned adhesion layers are formed on thetransparent substrate or the second substrate. Operation 808 is the sameas operation 605 of FIG. 6, with an additional choice of substrate. Insome embodiments, the patterned adhesion layer is formed on bothsubstrates. In FIG. 9E, the patterned adhesion layer 903 is formed onthe second substrate 901.

Referring back to FIG. 8, in operation 809 the transparent substrate isbonded to the second substrate via the patterned adhesion layer, asshown in FIG. 9F. While the patterned adhesion layer may be on thetransparent substrate or the second substrate, this operation is thesame as operation 609. In operation 811, the carrier wafer is removed.Operation 811 is the same as operation 611 of FIG. 6A. Optionally inoperation 813, the transparent substrate is grinded as in operations 221and 407. Finally, bio-material may be flowed in the biochip and attachedas in FIG. 9G.

One aspect of the present disclosure pertains to a method ofmanufacturing a biochip that includes forming first landings on a firstsubstrate, depositing a passivating layer over and between the firstlandings on the first substrate, depositing and planarizing an oxidelayer over the passivating layer on the first substrate, fusion bondingthe oxide layer on the first substrate to a transparent substrate,etching a backside of the first substrate in a pattern to expose some ofthe first landings, forming second landings on a second substrate,depositing a protective layer over the second landings on the secondsubstrate, forming through-holes in the second substrate, removing theprotective layer, and fusion bonding the first substrate and the secondsubstrate. The method may also include flowing adhesion layer material,support media material, and bio-material sequentially through thethrough-holes and fluidic channels to attach them to the first andsecond landings.

Another aspect of the present disclosure pertains to a method ofmanufacturing a biochip. The method includes forming first landings on afirst substrate, depositing a passivating layer over and between theplurality of first landings on the first substrate, depositing andplanarizing an oxide layer over the passivating layer on the firstsubstrate, fusion bonding the oxide layer on the first substrate to atransparent substrate, grinding the transparent substrate, bonding thetransparent substrate to a carrier wafer, etching a backside of thefirst substrate in a pattern to expose some of the plurality of firstlandings, depositing a protective layer over the exposed plurality offirst landings and the first substrate, forming through-holes in thetransparent substrate, removing the protective layer, fusion bonding thefirst substrate and a second substrate having a plurality of secondlandings thereon, and removing the carrier wafer. The method may alsoinclude flowing adhesion layer material, support media material, andbio-material sequentially through the through-holes and fluidic channelsto attach them to the first and second landings.

In yet another aspect, the present disclosure pertains to a method ofmaking a biochip. The method includes providing a transparent substrate,bonding a carrier wafer to the transparent substrate, forming apatterned passivating layer and first landings on the transparentsubstrate, forming a patterned adhesion layer on the transparentsubstrate, providing a second substrate having second landings andthrough-holes, bonding the transparent substrate to the second substratevia the patterned adhesion layer, and removing the carrier wafer. Themethod may also include flowing adhesion layer material, support mediamaterial, and biomaterial sequentially through the through-holes andfluidic channels to attach them to the first and second landings. Thebio-materials adhere to the first and second landings.

In some aspects, the present disclosure pertains to a method of making abiochip. The method includes providing a transparent substrate and asecond substrate, bonding a carrier wafer to the transparent substrate,forming a patterned passivating layer and first landings on thetransparent substrate, forming second landings on the second substrate,forming a patterned adhesion layer on the transparent substrate or thesecond substrate, forming through-holes in the transparent substrate,bonding the transparent substrate to the second substrate via thepatterned adhesion layer, and removing the carrier wafer. The method mayalso include flowing adhesion layer material, support media material,and bio-material sequentially through the through-holes and fluidicchannels to attach them to the first and second landings.

The present disclosure also pertains to a biochip having a transparentsubstrate and a bottom substrate, which may or may not be transparent.Microfluidic channels are disposed between the transparent substrate andthe bottom substrate. The sidewalls of the microfluidic channels may beformed of a first substrate different from the bottom substrate orformed of a patterned adhesion layer. The top, bottom, or both top andbottom of the microfluidic channels include first landings and secondlandings configured to bond to bio-materials which attach to thelandings after the biochip is formed.

In describing one or more of these embodiments, the present disclosuremay offer several advantages over prior art devices. In the discussionof the advantages or benefits that follows it should be noted that thesebenefits and/or results may be present is some embodiments, but are notrequired in every embodiment. Further, it is understood that differentembodiments disclosed herein offer different features and advantages,and that various changes, substitutions and alterations may be madewithout departing from the spirit and scope of the present disclosure.

What is claimed is:
 1. A biochip comprising: a transparent substrate; apatterned passivating layer on the transparent substrate; a patternedadhesion layer on the transparent substrate; a second substrate bondedto the transparent substrate, the second substrate having a plurality offirst landings, wherein the biochip comprises a microfluidic channelhaving the patterned adhesion layer as a sidewall, wherein themicrofluidic channel comprises: a first opening extending fully throughthe second substrate, the first opening being surrounded by a materialof the second substrate; a second opening extending fully through thesecond substrate, the second opening being different from the firstopening and being surrounded by the material of the second substrate;and a connecting portion connecting the first opening and the secondopening, wherein each opening into the connecting portion is surroundedby the material of the second substrate in a top down view, wherein thefirst landings overlie an exposed portion of the transparent substrate.2. The biochip of claim 1, further comprising second landings embeddedwithin the patterned passivating layer.
 3. The biochip of claim 2,wherein the first landings have a first density and the second landingshave a second density different from the first density.
 4. The biochipof claim 2, wherein the first landings comprise a first material and thesecond landings comprise a second material different from the firstmaterial.
 5. The biochip of claim 1, wherein the second substrate has athickness of between about 100 nm and about 200 nm.
 6. The biochip ofclaim 1, wherein the first landings are non-organic.
 7. The biochip ofclaim 6, wherein the first landings comprise a metal.
 8. A biochipcomprising: a transparent substrate; a patterned passivating layer onthe transparent substrate; a patterned adhesion layer to the transparentsubstrate; a second substrate bonded to the transparent substrate, thesecond substrate having a plurality of first landings, wherein thebiochip comprises a microfluidic channel having the patterned adhesionlayer as a sidewall, wherein the second substrate is a sensing wafer,wherein the microfluidic channel comprises: a first opening extendingfully through the second substrate, the first opening being surroundedby a material of the second substrate; a second opening extending fullythrough the second substrate, the second opening being different fromthe first opening and being surrounded by the material of the secondsubstrate; and a connecting portion connecting the first opening and thesecond opening, wherein each opening into the connecting portion issurrounded by the material of the second substrate in a top down view,wherein the first landings overlie an exposed portion of the transparentsubstrate.
 9. The biochip of claim 8, wherein the sensing wafer furthercomprises a temperature sensor.
 10. The biochip of claim 9, wherein thesensing wafer further comprises a heater.
 11. The biochip of claim 10,further comprising a pump.
 12. The biochip of claim 11, furthercomprising an embedded sensor.
 13. The biochip of claim 12, wherein theembedded sensor is an optical sensor.
 14. A biochip comprising: atransparent substrate; a patterned passivating layer on the transparentsubstrate; a patterned adhesion layer on the transparent substrate; asecond substrate bonded to the transparent substrate, the secondsubstrate having a plurality of first landings, wherein the biochipcomprises a microfluidic channel having the patterned adhesion layer asa sidewall, wherein the microfluidic channel comprises: a first openingextending fully through the second substrate, the first opening beingsurrounded by a material of the second substrate; a second openingextending fully through the second substrate, the second opening beingdifferent from the first opening and being surrounded by the material ofthe second substrate; a connecting portion connecting the first openingand the second opening, wherein each opening into the connecting portionis surrounded by the material of the second substrate in a top downview, wherein the first landings overlie an exposed portion of thetransparent substrate; and second landings located on the secondsubstrate.
 15. The biochip of claim 14, wherein the first landings havea first density and the second landings have a second density differentfrom the first density.
 16. The biochip of claim 15, wherein the firstlandings and the second landings are different materials.
 17. Thebiochip of claim 14, further comprising a flow meter.
 18. The biochip ofclaim 14, further comprising a pressure transducer.
 19. The biochip ofclaim 14, further comprising a heater.
 20. The biochip of claim 14,further comprising a pump.